Quantitative RT-PCR for Gene Expression

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed as previously described [10 (link)]. Briefly, RNA was isolated from cell cultures using TRIzol^® reagent according to the supplier’s instruction (Thermo Fisher,). RNA quality and quantity were determined using a Nanodrop 2000c UV-vis spectrophotometer (Thermo Fisher). cDNA was synthesized from 2.5 µg RNA using random nanomers and M-MLV reverse transcriptase (Invitrogen). Taqman primers and probes were designed using Primer Express 3.0.1 and are shown in Supplementary Table S1. All target genes were amplified using the Q-PCR core kit master mix (Eurogentec, Seraing, Belgium) on a 7900HT Fast Real-Time PCR system (Thermo Fisher). SDSV2.4.1 (Thermo Fisher) software was used to analyze the data. Expression of genes is presented in 2-delta CT and normalized to 18S.